Figure captions
Figure 1. Schematic illustration depicting cytosolic protein delivery using ultrasound-guided vaporization of phase-change nano-droplets. (A) The chemical structure of the protein anchoring reagent that anchors the protein onto the nano-droplets (DPPE-PEG-PyDTP) and the corresponding anchoring reaction. (B) Chemical structures of the nano-droplet modifiers for antibody modification (DPPE-PEG-Biotin), stabilization (DPPE-PEG-OMe), and labeling (DPPE-PEG-TAMRA). (C) Schematic of cellular uptake and ultrasound-guided intracellular vaporization of the droplets carrying cargo proteins for cytosolic delivery through endosomal escape.
Figure 2. Characterization of droplets for cytoplasmic protein delivery. (A) The concentration of the released β-galactosidase from the droplets after incubation with various concentrations of reduced glutathione (GSH). (B) Dynamic light scattering intensity of the droplets after down-sizing using an extruder. (C) Confocal microscopic images of DLD1 cells treated with nano-droplets modified with anti-epiregulin antibody and (D) without anti-epiregulin antibody. The antibody was labeled with a fluorescent dye (Alexa Fluor 647: AF647) (pink). The nano-droplets were labeled with DPPE-PEG-TAMRA (orange). Scale bar: 10 μm.
Figure 3. Fluorescence confocal microscopic images of cells after ultrasound-induced cytosolic delivery of β-galactosidase (β-Gal). (A) DLD1 cells treated with phase-change nano-droplets (PCNDs) carrying β-Gal and ultrasound, (B) those treated with β-Gal-carrying PCNDs without ultrasound, and (C) those treated with PCNDs not carrying β-Gal and ultrasound were observed after staining with a fluorescent substrate for β-Gal (TokyoGreen-βGal) and a endosomal and lysosomal staining dye (Lysotracker). (D) Intact cells were also observed as the control in the same way. The fluorescence images for TokyoGreen-βGal (green) and Lysotracker (cyan) were merged along with the differential interference contrast images. Scale bar: 25 μm.
Figure 4. Viabilities of cells after ultrasound (US)-induced cytosolic delivery of saporin (Sap). DLD1 cells were treated with the nano-droplets (PCNDs) with or without Sap and then were exposed to ultrasound (US) or not. In the same way, the viability of intact cells was examined. The viability was normalized to that of the intact cells. Each bar represents the mean ± S.E. (n = 3). *p < 0.01 and **p < 0.05 versus the cells treated with both PCNDs carrying Sap and US (t-test).